Ziegelmüller Jana, Kouvelas Nikolaos, Schwaller Nino, Thambythurai Priyanka, Hofer Alexander M, Mühlemann Oliver, Karousis Evangelos D
Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland.
Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
J Biol Chem. 2025 May 28;301(7):110307. doi: 10.1016/j.jbc.2025.110307.
Cell-free translation systems are indispensable for studying protein synthesis, enabling researchers to explore translational regulation across different cell types. The difficulties in producing cell-free translation systems from different cell types limit the ability to study regulatory mechanisms that depend on different biological contexts. Here, we present a scalable method for preparing translation-competent lysates from a range of frequently used human cell lines using dual centrifugation. We optimized lysis conditions for adherent and suspension cells, producing high-quality lysates from HEK-293 (adherent and in suspension), HeLa, SH-SY5Y, and U2OS cells. Our results demonstrate that cell-specific factors influence translation efficiency, with adherent HeLa cells showing the highest activity. We also observed that sensitivity to lysis conditions varies between cell lines, underscoring the importance of fine-tuning parameters for efficient protein production. Our method provides a robust and adaptable approach for generating cell-type-specific lysates, broadening the application of in vitro translation systems in studying translational mechanisms.
无细胞翻译系统对于研究蛋白质合成至关重要，使研究人员能够探索不同细胞类型中的翻译调控。从不同细胞类型制备无细胞翻译系统存在困难，限制了研究依赖于不同生物学背景的调控机制的能力。在此，我们展示了一种可扩展的方法，通过双重离心从一系列常用的人类细胞系中制备具有翻译活性的裂解物。我们优化了贴壁细胞和悬浮细胞的裂解条件，从HEK-293（贴壁和悬浮）、HeLa、SH-SY5Y和U2OS细胞中产生了高质量的裂解物。我们的结果表明，细胞特异性因子会影响翻译效率，贴壁的HeLa细胞表现出最高的活性。我们还观察到不同细胞系对裂解条件的敏感性不同，这突出了微调参数以实现高效蛋白质生产的重要性。我们的方法为生成细胞类型特异性裂解物提供了一种强大且适应性强的方法，拓宽了体外翻译系统在研究翻译机制中的应用。
